The manipulation of cells via introduction of exogenous materials serves as a critical enabler for a broad spectrum of applications, including drug discovery, transgenics, and cell-based therapeutics. However, in many applications progress is nevertheless constrained by the limitations of current manipulation techniques. The long-term goal of this proposal is to address this issue through development of advanced instrumentation for microinjection-based manipulation. Although microinjection represents the "gold standard" for cellular manipulation, it has been largely relegated to niche applications by its labor-intensiveness and low throughput (~3 cells/min), which result from reliance upon skilled operators and serialized injection methodologies. The objective of this proposal is to develop ultrahigh throughput (UHT) microinjection instrumentation that addresses these limitations through automation, massive parallelization, and monolithic integration. This instrumentation will be based upon a microelectromechanical systems (MEMS) device core, composed of a massively parallel array of cell Capture Sites with monolithically integrated Injectors, which will enable simultaneous capture and injection of many thousands of cells/min with minimal need for human or robotic involvement. Guided by preliminary studies demonstrating feasibility of the first functional element of this instrumentation, namely UHT cell capture, the proposed effort seeks to take the next steps forward through pursuit of a staged research plan that gradually introduces additional functionalities. The Specific Aims are: 1) Develop prototype for UHT cell capture and permeabilization;and 2) Develop prototype for UHT microinjection. Novel microfabrication processes will be developed for the MEMS device cores and computer-controlled external subsystems will be developed that add high-speed cell handling functionality. Instrument functionality will then be validated using live cell testing (i.e. capture, injection, release, and transfection efficiency, as well as viability). The proposed research is innovative because it represents the first attempt to address microinjection's limitations through not only automation, but also massive parallelization and monolithic integration of all functionality into a single MEMS device. This research is significant because it may: a) simplify microinjection sufficiently to make it accessible to a broader range of researchers;b) considerably enhance current applications where throughput is often a limiting factor, e.g. transgenics;and c) serve as a fundamental enabler for applications where progress is constrained by safety or efficacy concerns associated with current manipulation techniques, e.g. ex vivo cell therapies based on genetic modification. PUBLIC HEALTH RELEVANCE (provided by applicant): The proposed effort seeks to develop innovative instrumentation for cellular manipulation that holds potential for opening new avenues of biological investigation at larger scales than previously possible across many disciplines. It may also enable realization of novel techniques for curing genetic diseases and engineering cellular therapies for other diseases. As such, the relevance of the proposed effort to NIH's mission to advance understanding of biological systems, improve control of disease, and enhance health is apparent.